Carbohydrate binding molecules

ABSTRACT

The present invention provides compounds, compositions, medicaments and methods comprising or using carbohydrate binding molecules. More specifically, the invention provides a means of treating diseases and/or conditions caused or contributed to by pathogens, particularly microbial pathogens and methods of screening, identifying, detecting tagging and/or labelling carbohydrates.

FIELD OF THE INVENTION

The present invention provides compounds, compositions, medicaments andmethods comprising or using carbohydrate binding molecules. Morespecifically, the invention provides a means of treating diseases and/orconditions caused or contributed to by pathogens, particularly microbialpathogens and methods of screening, identifying, detecting taggingand/or labelling carbohydrates.

BACKGROUND OF THE INVENTION

The surfaces of most mammalian cells are rich in glycoconjugates andmany pathogens have exploited the terminal carbohydrate of theseglycoconjugates for cell attachment during the initial stages ofpathogenesis. By way of example, the viral pathogens influenza andparainfluenza both bind to sialic acid receptors at the mammalian cellsurface.

Sialic acid recognition is mediated via lectins or lectin-like moleculesand their corresponding receptors (3). In most cases, sialic acidbinding lectins, which also include several viral glycoproteins andbacterial toxins (2,4), as well as the mammalian lectin superfamiliessuch as the siglecs (5) and selectins (6), bind to their receptor withrelatively high affinity due to the multivalent nature of thesemolecules, thus alleviating the low intrinsic affinity that mostprotein-carbohydrate interactions are associated with (7,8). Generally,association constants (K_(a)) for the binding of monovalent and divalentsialosides by such lectins can reach 10⁴M⁻¹. However, by virtue of theirmultivalency, some sialic acid binding lectins can interact withmultivalent cell surface glycans to achieve affinities reaching 10⁹M⁻¹by an avidity effect. These enhanced affinities have been shown in partto be due to improved structural packing of proteins promoted by ligandbinding, associated with favourable binding energetics (9-11). One ofthe best-studied multivalent lectin-sialic acid interactions is theinfluenza virus trimeric hemagglutinin, which can achieve affinities upto 10⁸M⁻¹ compared to around 4×10²M⁻¹ when one, or both of the entitiesare not in a multivalent state (12).

Sialidases, or neuraminidases, catalyze the hydrolysis of sialic acidsfrom a variety of glycoconjugates and are often modular enzymes,containing accessory modules attached to the catalytic core of theprotein. Some of these modules have been identified as carbohydratebinding modules (CBMs). CBMs are found widely in glycoside hydrolasesand are discrete, non-catalytic modules that primarily exist to targetthe parent enzyme to its substrate for efficient hydrolysis byincreasing the concentration of the enzyme at the substrate surface(13). The modules can be single, tandem or in multiples within theglycosyl hydrolase architecture. Studies have shown that they can bindto their specific glycans independently when isolated from the parentmolecule, and can behave in a cooperative manner when isolated in tandem(14,15). Currently, CBMs are grouped into 52 families based upon primarysequence similarity (http://www.cazy.org/fam/acc_CBM.html). Subtledifferences in the structures of CBMs can lead to diverse ligandspecificity, which make CBMs an attractive system for eludicatingprotein-carbohydrate mechanisms.

A poster entitled “Engineering Multivalent Sialic Acid Recognition usingthe CBM40 module from Vibrio cholerae Sialidase” (published 17 May 2008:seehttp://www.biochem.emory.edu/conferences/glycot/Images/GlycoTProgram-Posters.pdf)describes the development of reagents with increased affinity for sialicacid through multivalency. However, the poster does not disclose thatsuch reagents have any application in the treatment of diseases and/orconditions caused by pathogens.

It is well documented that there is increasing resistance to currentlyavailable influenza antivirals (in particular, Roche's Tamiflu) and thishas emphasized the need to look at alternative methods to combatinfluenza. Previous studies have indicated the use of non-toxic lectins,such as SNA lectin from the elderberry as an influenza virus inhibitorbut this demonstrated weak binding to sialic acid and required thepresence of two or three different sugar moieties for recognition andeffective inhibition. Recent work using a recombinant sialidase-fusionprotein designated DAS181 (Fludase, developed by NexBio Inc.) iscurrently being investigated as another of these alternatives. Thisprotein effectively removes sialic acids from the surface of epithelialcells, rendering the virus unable to bind to receptors. However, byremoving sialic acids using a sialidase, this can also expose crypticreceptors, which may serve as receptors for other pathogens.

The present invention aims to provide compounds, compositions,medicaments and methods useful in the treatment of diseases and/orconditions caused by pathogens and to obviate the problems associatedwith the prior art.

SUMMARY OF THE INVENTION

The present invention is based on the finding that carbohydrate-bindingmolecules (CBMs) may be exploited to combat and/or prevent diseases orconditions, which occur as a result of infection with viral and/orbacterial pathogens.

It is known that in order to bind/adhere, colonise or gain entry intocells, a number of pathogens exploit the presence of carbohydrates oncell surfaces. By way of example, respiratory pathogens such as virusesbelonging to the Orthomyxoviridae or Paramyxovirus families utilise cellsurface carbohydrates to bind and gain entry to specific cell types in avariety of mammalian tissues. Similarly, bacteria such as thosebelonging to the Streptococcus genus exploit cell surface carbohydratesas a means of binding/adhering to and/or entering certain cells.

The surfaces of mammalian cells comprise numerous different types ofmolecule. In particular, mammalian cells are rich in glycoconjugates,the terminal carbohydrate of which is typically sialic acid.Accordingly, certain pathogens have evolved to exploit the presence ofsialic acid at the cell surface to bind/adhere and/or gain entry tothose cells.

Members of the CBM group normally function to target or direct glycosylhydrolase enzymes such as sialidase or neuraminidase, to theirsubstrates for efficient hydrolosis. As such, CBMs have an affinity forcell surface carbohydrates such as, for example, sialic acid, galactose,fucose, N-acetylglucosamine, and blood group antigens. One of skill inthe art will appreciate that a compound which exhibits an affinity for acell surface carbohydrate, may be used to block a pathogen's ability tobind to, or recognise, that carbohydrate and thus may prevent thepathogen from colonising and/or entering the cell.

Accordingly, the inventors have discovered that by exploiting the CBM'saffinity for carbohydrates, it is possible to provide compounds,compositions, medicaments and/or methods useful in the treatment of arange of diseases and/or conditions caused or contributed to by thosepathogens which bind/adhere or otherwise associate with cell surfacecarbohydrates during pathogenesis.

Thus, in a first aspect, the present invention provides acarbohydrate-binding module (CBM) for use in treating or preventing adisease and/or condition caused or contributed to by one or morepathogens.

In a second aspect, the present invention provides the use of acarbohydrate-binding module (CBM) for the manufacture of a medicamentfor the treatment or prevention of a disease and/or condition caused orcontributed to by one or more pathogens.

In addition to the above, a third aspect of this invention provides apharmaceutical composition comprising a CBM for use in treating orpreventing a disease and/or condition caused or contributed to by one ormore pathogens.

Preferably, the pharmaceutical compositions provided by this inventionare formulated as sterile pharmaceutical compositions and suitableexcipients, carriers or diluents may include, for example, water,saline, phosphate buffered saline, dextrose, glycerol, ethanol, ionexchangers, alumina, aluminium stearate, lecithin, serum proteins, suchas serum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water salts or electrolytes, such as protaminesulphate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycon,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polypropylene-block polymers, polyethylene glycol and woolfat and the like, or combinations thereof.

Said pharmaceutical composition may be formulated, for example, in aform suitable for oral, parenteral or topical administration.

One of skill in the art will appreciate that in addition to the use ofindividual or single CBMs which bind specific carbohydrates, thecompounds, compositions and medicaments provided herein may furthercomprise other CBMs which either bind to or express an affinity for, analternate carbohydrate, or which exhibit a different affinity for thesame carbohydrate.

As stated, in addition to providing compounds, compositions and/ormedicaments for use in treating or preventing a variety of diseases andor conditions, the present invention also provides methods useful in thetreatment of subjects, particularly human subjects, suffering from, orat risk of contracting, a disease and/or condition caused or contributedto by a pathogen, particularly those pathogens which are capable ofbinding cell surface carbohydrates.

Accordingly, a fourth aspect of this invention provides a method oftreating or preventing a disease caused or contributed to by a pathogen,said method comprising the step of administering a therapeuticallyeffective amount of a carbohydrate binding module (CBM).

The present invention represents a distinct advantage over existingtreatments for diseases and/or conditions caused or contributed to bypathogens (for example viral and/or bacterial respiratory pathogens)which involve, for example, the enzymatic removal of cell surfacecarbohydrates. Strategies of this type may expose cryptic receptors toother pathogens rendering the cells vulnerable to infection. By blockingaccess to cell surface carbohydrates rather than removing them from thecell surface, this problem is avoided and furthermore, since the CBMsdescribed herein bind to terminal carbohydrate residues (such as, forexample sialic acid), the CBM might not elicit an immune response.

The term “pathogen” as used herein, should be understood as encompassingany pathogen which which is capable of binding, recognising or otherwiseassociating with a cell surface carbohydrate and particularly thosewhich have evolved to exploit or utilise the presence of cell surfacecarbohydrates as a means of binding/adhering to and/or entering a cell.As such, in one embodiment, the term “pathogen” encompasses microbialpathogens and includes respiratory pathogens such as viruses belongingto the Orthomyxoviridae or Paramyxoviridae families, for exampleinfluenza and human parainfluenza, as well as bacteria belonging to theStreptococcus genus such as Streptococcus pneumoniae and/or Hemophilusinfluenzae, Pseudomonas aeruginosa—all of which are capable of bindingcarbohydrates on the surface of mammalian cells. One of skill willappreciate that the mammalian cell most frequently colonised/infected bypathogens of the type described herein, are epithelial cells, especiallythose lining the mucosal tracts (for example, respiratory epithelialcells).

In view of the above, the diseases and/or conditions treatable orpreventable using the compounds, compositions, medicaments or methodsprovided herein are many and varied and should be considered as thosewhich are caused or contributed to by any of the pathogens mentionedherein. Accordingly, diseases and/or conditions caused or contributed toby viral respiratory pathogens such as, for example Influenza and/orParainfluenza and bacterial pathogens such as, for example,Streptococcus pneumoniae, may benefit from the use of the variouscompounds, compositions, medicaments and/or methods provided herein. Inthis regard, the present invention provides compounds, compositions,medicaments and/or methods for use in treating and/or preventingdiseases or conditions such as influenza and viral and/or bacterialrespiratory diseases such as croup, pneumonia and bronchitis. In view ofthe above, one embodiment of the present invention provides compounds,compositions, medicaments and/or methods for use in treating diseasescaused or contributed to by one or more pathogens selected from thegroup consisting of Influenza virus; Parainfluenza virus; andStreptococcus pneumoniae.

The term “carbohydrate” should be understood to encompass anycarbohydrates especially those present at the cell surface and inparticular those present on the surface of mammalian cells, such asmammalian epithelial cells. It should also be understood that while theterm “carbohydrate” may encompass monosaccharide units, it may alsoencompass polysaccharides, glycan chains and or the carbohydrate moietyof a glycoconjugate. As such, the term “carbohydrate” may include, forexample, the sialic acids, a family of carbohydrates having anine-carbon backbone and which are located at the terminal (or distal)end of glycan chains at the cell surface. The sialic acid familyencompasses a number (approximately 50) of derivatives that may resultfrom acetylation, glycolylation, lactonization and methylation at C4,C5, C7, C8 and C9. Furthermore, sialic acids are found linked α(2,3) orα(2,6) to Gal and GalNAc or α(2,8) or α(2,9) to another sialic acid.Accordingly, it is important to understand that while the term “sialicacid” is used throughout this specification, it should be understood asencompassing all derivatives, analogues or variants (either naturallyoccurring or synthetically generated) thereof as well as dimers,trimers, oligomers, polymers or concatamers comprising the same. Othercell surface carbohydrates could include N-acetylglucosamine, galactose,N-acetylgalactosamine, fucose, mannose, and their complexes found insuch epitopes as blood group antigens.

CBMs are found widely in nature and exhibit a wide range of affinitiesfor a variety of different carbohydrates. To date, the CBMs have beengrouped into 52 families based on primary sequence similarity and itshould be understood that the invention may concern any CBM. Of course,in order to treat a disease or condition known to be caused by apathogen, which binds to a particular cell surface carbohydrate, a CBMwhich binds to or exhibits an affinity for that carbohydrate, will beparticularly useful in the treatment or prevention of that disease orcondition.

One embodiment of the invention concerns those CBM molecules, whichexhibit an affinity for or bind to sialic acid. One of skill willappreciate that CBMs which bind to or exhibit an affinity for sialicacid, will be useful as compounds or in compositions, medicaments ormethods for treating diseases and/or conditions caused or contributed toby pathogens which bind or otherwise associate with sialic acid duringpathogenesis.

It should be noted that a number of different CBMs may exhibit anaffinity for a particular carbohydrate and the magnitude of thataffinity may vary. Nevertheless, it should be understood that in thecase of CBMs which bind cell surface carbohydrates such as sialic acid,any CBM exhibiting an affinity for sialic acid should be considered aspotentially useful.

Among those potentially useful CBMs are those which belong to family 40and which may otherwise be known as the “CBM40” CBMs. Included withinthe CBM40 family are CBMs derived from Vibrio cholerae and Clostridiumperfringens.

The CBM40 derived from V. cholerae exhibits a relatively high affinity(K_(d)˜30 μM) for sialic acid whereas the CBM40 derived from C.perfringens exhibits a high millimolar affinity towards sialic acid.

In addition to the above, the inventors have discovered that while asingle CBM molecule may exhibit an affinity for a particularcarbohydrate (such as, for example, sialic acid), by linking orcombining together two or more CBM molecules, it is possible, throughavidity, to generate a molecule having a higher affinity for thatcarbohydrate.

Thus, this invention also concerns multivalent CBM molecules orpolypeptides referred to hereinafter as “CBM polymers”, which compriseone or more CBMs (or monomers) joined, linked or conjugated together bysome means.

Accordingly, a fifth aspect of this invention provides a CBM polymercomprising two or more CBMs (CBM monomers).

The term “polymer” will be readily understood by one of skill in thisfield to encompass molecules comprising a number of linked units. Inthis regard, the term “CBM polymer” may be used to describe CBM-dimers(i.e. two CBM monomers), CBM-trimers (three CBM monomers), CBM-tetramers(four CBM monomers) and larger, CBM-oligomers (five or more CBMmonomers).

One of skill in the art will appreciate that although it may bedesirable to generate CBM polymers comprising identical repeating CBMmonomers (for example repeating V. cholerae derived CBM40 monomers), itmay also be possible to create CBM polymers which comprise a number ofdifferent CBM monomers, each being capable of binding a differentcarbohydrate and/or each exhibiting a different affinity for aparticular carbohydrate. Similarly, CBM polymers, which comprise CBMmonomers binding, or exhibiting an affinity for, different cell surfacecarbohydrates are also within the scope of his invention. As such, a CBMpolymer according to this invention may comprise CBM monomers havingaffinity for sialic acid and CBM monomers having affinity for or bindingto some other carbohydrate, for example mannose, galactose, fucose,N-acetylglucosamine.

As stated, by joining, linking or conjugating together CBM monomers, itis possible to create a larger polymeric molecule, which, throughavidity, exhibits a greater affinity for a particular carbohydrate orfor a number of carbohydrates. Thus, while a particular CBM monomer, forexample the CBM40 derived from V. cholerae may exhibit affinity forsialic acid, when two or more of said CBM40 monomers are linked, joinedor conjugated together, the resulting polymer may exhibit an increasedaffinity for sialic acid.

Accordingly, the invention and in particular the term “CBM” as used inthe first, second and third and fourth aspects of this invention, shouldbe understood as including the CBM polymers described herein.

One of skill in the art will appreciate that a CBM polymer comprisingCBM monomers, which bind to or have affinity for differentcarbohydrates, may itself bind to or exhibit an affinity for the samecarbohydrates. For example, if a CBM polymer comprises CBM monomerswhich bind to or have affinity for two different cell surfacecarbohydrates, the resulting CBM polymer may also bind to or exhibit anaffinity for the same two cell surface carbohydrates.

CBM polymers which comprise a number of different CBM monomers andconsequently bind to or exhibit an affinity toward a number of differentcarbohydrate molecules, may be particularly useful in compositions,compounds, medicaments and/or methods to treat or prevent diseasesand/or conditions caused or contributed to by more than one pathogen.

For example, where a subject suffers from or is predisposed tocontracting at least two diseases or conditions caused or contributed toby two different pathogens each of which binds a different cell surfacecarbohydrate (as a means of colonising and/or entering cells) a CBMpolymer comprising CBM monomers which recognise, bind or otherwiseassociate with these carbohydrates may be useful in the treatment orprevention of said diseases and/or conditions. Alternatively, diseasesand/or conditions caused by two or more pathogens may be treated usingcompounds or compositions, medicaments or methods utilising the relevantmonomeric CBMs.

One of skill will appreciate that there are many ways in which aminoacids, proteins and/or peptides can be linked together. In oneembodiment, amino acids, peptides and/or proteins may be joined linkedor conjugated by chemical means and/or via cloning and/or PCRtechnology.

By way of example, the nucleotide sequence encoding the relevant CBM(s)may be amplified by PCR and modified such that one or more copies of thesame can be ligated together or to other CBM encoding nucleotidesequences. In addition, the CBM nucleotide sequences may be furthermodified to include at their 3′ and/or 5′ ends sequences encoding linkermoieties comprising one or more amino acid residues.

CBM polymers (or multivalent CBMs) may also be generated using CBMmonomers modified to include, for example, oligomerisation domains.Molecules possessing such domains may self assemble to form oligomericstructures. In one embodiment the oligomerisation domain may be derivedfrom, for example bacterial species such as Pseudomonas aeruginosa whichis known to encode a trimerisation domain. PCR based techniques may beused to modify CBM molecules in this way.

The amplified and optionally modified/ligated CBM sequences may then becloned into suitable vectors for expression and purification of theresulting CBM or CBM polymer. Recombinant CBM monomers (such as thoseencoding oligomerisation domains) or CBM polymers, may be expressed in,for example, E. coli.

In view of the above, a sixth aspect of this invention may provide amethod of generating a CBM polymer, said method comprising the step of:

-   -   (a) ligating CBM encoding nucleotide sequences; and    -   (b) expressing the ligated CBM nucleotide sequences to generate        a CBM polymer.

As stated, the CBM polymers provided by this invention may comprise CBMmonomers linked by some form of linker moiety, which may take the formof one or more amino acids. Although the precise number of amino acidscomprising the linker moiety may vary, typically, the linker moiety willcomprise any length from 1 to about 30 amino acids.

An alternative means of generating a CBM polymer is to link, throughmolecular biological techniques, an oligomerisation domain to the CBM.For example, the trimerisation domain found in the pseudaminidase ofPseudomonas aeruginosa (residues 335 to 438, Xu, G., Ryan, C., Kiefel,M. J., Wilson, J. C. and Taylor, G. L. (2009) J. Mol. Biol. 386(3),828-840) could be linked via a linker peptide to one or more CBM(s).Expression of a single construct would then generate a trimer withincreased affinity through an avidity effect. Similarly, thetetramerisation domain from the human vasodilator-stimulatedphosphoprotein (Kuhnel K., et al. PNAS 2004, 101, 17027-17032), a45-residue peptide which forms a tetrameric coiled-coil structure couldbe linked, via a suitable linker, to one or more CBM(s), to form, forexample, a tetrameric oligomer with increased affinity through anavidity effect.

A seventh aspect of this invention provides a method of screening for oridentifying CBMs potentially useful in the treatment of diseases and/orconditions, said method comprising the steps of:

-   -   (i) contacting a CBM or CBM polymer with a cell in the presence        of a pathogen known to bind or infect said cell;    -   (ii) identifying those cells to which the pathogen has bound or        which the pathogen has infected; and    -   (iii) comparing the results with a standard or control assay in        which no CBM or CBM polymer has been added;

wherein a decrease in the number of cells to which the pathogen hasbound or a decrease in the number of cells infected by the pathogenrelative to the control assay, indicates a CBM or CBM polymerpotentially useful in the treatment of a disease and/or condition causedor contributed to by that pathogen.

The cell contacted with the CBM/CBM polymer may take the form of a cellmonolayer cultured under tissue culture conditions or a tissue biopsy orscraping obtained from a subject. In addition, the cell may be presentin an animal, such as a rodent (mouse, rat, guinea pig, rabbit or thelike). Where an animal is used in the methods provided by the seventhaspect of this invention, the animal may be administered the CBM or CBMpolymer to be tested and simultaneously (or before or after) infectedwith a pathogen (for example a respiratory pathogen). Furthermore, thestandard or control assay may take the form of an animal, which has beeninfected with a pathogen but not administered the CBM or CBM polymer.

When using animals, in order to determine whether or not a CBM or CBMpolymer is potentially useful in the treatment or prevention of adisease caused or contributed to by a particular pathogen, aftercompletion of the method provided by the seventh aspect, cells may bederived from the animal and examined to determine whether or not thepathogen has bound thereto or infected the cells. Additionally oralternatively, the animal may be observed for signs or symptoms ofdisease. When compared to an animal subjected to the control or standardprotocol, a reduction in the severity of the symptoms may indicate thatthe CBM or CBM polymer administered to that animal might be useful inthe treatment of diseases and or conditions caused or contributed to bythat pathogen.

In addition to the above, the inventors have discovered that thedifferential binding capabilities of the various CBMs and CBM polymersdescribed herein, can be exploited to provide a means of screening,identifying, detecting, labelling and/or tagging carbohydrates.

Thus, in an eighth aspect, the present invention provides a method ofscreening, identifying, detecting, labelling and/or tagging acarbohydrate in a sample, said method comprising the step of:

-   -   (a) contacting a sample with a CBM or CBM polymer as described        herein, under conditions suitable to permit binding between the        CBM/CBM polymer and the carbohydrate(s) it/they bind or have        affinity for;    -   (b) removing unbound CBM or CBM polymer; and    -   (c) detecting bound CBM or CBM polymer.

One of skill in the art will appreciate that since CBMs exhibit a degreeof specificity for certain carbohydrates, the CBM or CBM polymerdetected in step (c) will serve as an indication of the carbohydratespresent in the sample.

It should be understood that the term “sample” encompasses biologicalsamples such as bodily fluids (urine, blood, plasma, serum, sweat,saliva, semen and the like) as well as samples derived from othersources such as, for example, food, beverages and water sources (rivers,oceans). Indeed, almost any sample thought to contain carbohydrates andto which CBM or CBM polymer can be added, may be used.

In one embodiment, the sample comprises cells, preferably mammaliancells, derived from a tissue biopsy, scraping or secretion. In this way,the method provided by the eighth aspect of this invention may be usedto detect the presence of certain cells in a sample. One of skill inthis field will readily understand that because cells express a range ofcarbohydrates at their surface, by contacting a sample with a CBMspecific to a carbohydrate known to be expressed on a certain cell typeand detecting whether or not the CBM has bound to any cell in thesample, it may be possible to identify the presence of certain cellswithin a heterogeneous cell population. Techniques such as FACS may beused to identify cells labelled or tagged with CBMs or CBM polymers(optionally modified to include a detectable tag) according to thisinvention. In addition techniques of this type may be useful fordiagnosing diseases or other conditions. For example, CBMs or CBMpolymers which bind to or have affinity for carbohydrates known to beexpressed on cancerous cells may be used to identify or detect thepresence of cancerous cells in a sample.

In order to remove unbound CBM or CBM polymer, the sample::CBM/CBMpolymer complexes resulting from step (a) above may be subjected to oneor more wash steps with an appropriate buffer.

In one embodiment, the sample may be immobilised on a suitable substratesuch as, for example, plastic, glass, agarose, nitrocellulose, paper orthe like.

One of skill in the art will appreciate that the CBMs or CBM polymersprovided by this invention may be further modified to include one ormore detectable tags or labels. In this way, modifying, or conjugatingthe CBM, or CBM polymer to include an enzyme capable of reporting alevel via a colormetric chemiluminescent reaction may achieve thedetection of bound CBM or CBM polymer. Such enzymes may include but arenot limited to Horse Radish Peroxidase (HRP) and Alkaline Phosphatase(AlkP). Additionally, or alternatively, the CBM or CBM polymers providedherein may be conjugated to a fluorescent molecule such as, for examplea GFP and fluorophore, such as FITC, rhodamine or Texas Red. Other typesof molecule, which may be conjugated to the CBM or CBM polymersdescribed herein, may include radiolabelled moieties.

In a further embodiment, the CBM polymers or CBMs provided by thisinvention may be further modified to include a moiety, for example anantibody, a small organic molecule a nucleic acid, drug or toxin, to bedelivered to a cell. By identifying those carbohydrates expressed at acell surface, a CBM or CBM polymer, which binds to or has affinity forone or more of the identified carbohydrates, may be used to deliver amoiety of the type listed above, to that cell.

DETAILED DESCRIPTION

The present invention will now be described in detail with reference tothe following Figures, which show:

FIG. 1: (A) Schematic drawing of the Vibrio cholerae sialidase showingthe central catalytic domain flanked by the lectin-likecarbohydrate-binding modules (CBMs). The sialic acid recognizing CBM,CBM40, is on the right. (B) Schematic view of the constructs made inthis study.

FIG. 2: Sialosides used in the ITC study. (A) 3′sialyllactose, (B)6′sialyllactose), (C) disialyllactose (DSL), (D)disialyl-lacto-N-tetraose (DSLNT).

FIG. 3: Crystal structure of V. cholerae CBM40 in complex with3′sialyllactose. (A) Interactions of the ligand with the CBM withhydrogen-bonds drawn as dashed lines. The sigma-a weighted Fo-Fcelectron density map is drawn at 3σ. (B) The relationship between thethree CBM40 modules in the asymmetric unit of the crystal.

FIG. 4: Isothermal titration calorimetry isotherms showing the bindingof various ligands to the isolated CBM40. (A) 3′SL, (B) 6′SL, (C) DSL,(D) DSLNT.

FIG. 5: Isothermal titration calorimetry isotherms showing the bindingof 3′SL to various CBM40 constructs. (A) 2CBM(5), (B) 2CBM(10), (C)2CBM(15), (D) 3CBM(5), (E) 3CBM(10), (F) 4CBM(5).

FIG. 6: Surface plasmon resonance (SPR) sensorgrams showing the bindingof the CBM40 modules to immobilised 3′sialyllactose receptors. (A) 1CBM,(B) 2CBM(5), (C) 2CBM(10), (D) 2CBM(15), (E) 3CBM(5), (F) 3CBM(10), (G)4CBM(5).

FIG. 7: Van't Hoff plot derived from the SPR experiments at differenttemperatures.

FIG. 8: Plaque assay demonstrating that multivalent CBM40 polypeptidescan reduce the binding of influenza virus in vitro.

FIG. 9: Hemagglutination assay of chicken red blood cells with (a)vaccine X31, monomeric and multivalent CBM40, and (b) with and withoutthe presence of influenza A virus from vaccine strain X31.

FIG. 10: Mammalian MDCK cell protection by 3CBM(5), 4CBM(5) and CBMTDpolypeptides against influenza strain A/Udorn/72 (H3N2).

FIG. 11: (A) GFP-3CBM40 (0.5 mg/ml) binding to cell surface of MDCKcells; (B) Phase contrast micrographs of MDCK cells incubated withGFP-3CBM40, 72 p.i with the Udorn virus. Panel (a) CBM (0.5 mg/ml) only,(b)-(f) 0.5, 0.1, 0.05, 0.01 and 0.005 mg/ml CBM with Udorn viruspresent, (g) Udorn virus only, and (h) uninfected cells.

FIG. 12: Effect of 3CBM40 on lung virus titre (A), and mouse weights (B)in influenza A virus infected BALB/c mice. Mice were administered with3CBM40 (black bars) or with BSA (white bars) 1 day before, on the day,or 1 or 3 days after H1N1 infection (10³ virus pfu). Data were collectedfrom 4 mice per group at 6 days p.i. The virus control group (Day 0) isindicated as PBS on the bar charts.

MATERIALS AND METHODS Recombinant DNA Techniques

The DNA fragment encoding the family 40 CBM of V. choleraesialidase/neuraminidase (VCNA), residues 25-216, was amplified by PCRfrom the construct pET30b+ containing the nanH gene (16) usingoligonucleotide primer pair 1F and 1R based on the sequence outline inTable 2. The amplified DNA fragment of 573 bp was digested with Nco Iand Xho I and cloned into the pEHISTEV vector (an engineered variant ofpET30 with an N-terminal 6× His tag that is cleaved by Tobacco EtchVirus (TEV) protease) (Dr H. Liu, unpublished work), digested with thesame enzymes and used to transform E. coli DH5a. The construct, p1CBMwas verified by DNA sequencing (University of Dundee Sequencing Service,UK).

The DNA fragment, encoding the V. cholerae CBM40, was modified at the5′- and 3′-termini to incorporate different restriction endonucleasesites through the use of different primer pairs. This was performed toallow ligation of individual copies of the DNA fragment to generate 2, 3and 4 copies in tandem (FIG. 1B). Moreover, the primer pairs allowed theinsertion of 5, 10 or 15 codons, to represent the length of amino acidslinking the individual modules. All these modifications were achieved byPCR amplification using different primer pairs outlined in Table 2, andp1CBM as template. The resulting fragments were cloned into anappropriately digested pEHISTEV vector until the desired number ofmodules was achieved. These were labelled p2CBM(5), p2CBM(10),p2CBM(15), p3CBM(5), and p3CBM(10), representing 2 and 3 repeatingsialic acid-binding domains with the number of amino acids in the linkerin parenthesis, respectively. For p4CBM(5), HindIII-EcoRI-modified andEcoRI-XhoI-modified DNA fragments were initially cloned into pET17bdigested with the appropriate enzymes before assembling the final genein pEHISTEV. All constructs were propagated in E. coli DH5a. Positiveclones were verified by DNA sequencing before transforming expressionhosts E. coli BL21(DE3) (1CBM, 2CBM(5), 3CBM(5), 3CBM(10), 4CBM(5) andE. coli BL21(DE3) Gold (2CBM(10), and 2CBM(15) respectively) for proteinproduction.

Protein Production and Purification

All constructs were grown and expressed as described for VCNA (16).Briefly, 1 L aliquots of Luria-Bertani Broth media containing 30 μg/mlkanamycin, were inoculated with single colonies and grown at 37° C.until the optical density of the culture reached 0.4-0.6 at 600 nm.Cells were subjected to heat shock for 20 min in a 42° C. water bathbefore being cooled down to 25° C. and induced with 1 mM IPTG, and leftto shake overnight at the same temperature. Cells were harvested bycentrifugation (8000×g, 4° C.), and pellets frozen at −20° C., untilrequired.

All polypeptides were purified by nickel affinity chromatography aspreviously described (16). Samples were analysed using SDS PAGE andpartially purified polypeptides were dialyzed into TEV protease cleavagebuffer (PBS, 0.3M NaCl, 1 mM DTT, 0.5 mM EDTA, 20 mM imidazole) anddigested overnight with TEV protease. Each polypeptide was then furtherdialysed with PBS, 0.3M NaCl, 10 mM imidazole buffer before a secondround of purification on a nickel-charged column to remove undigestedHis-tagged polypeptides. Untagged polypeptide samples were dialysedextensively in 10 mM HEPES buffer, pH 7.4, 0.15M NaCl and concentratedbefore use.

Crystallisation and Structure Determination

Crystals of the single V. cholerae CBM40 were obtained in 0.1M MOPS pH6.5, 1.1M lithium sulphate, 0.6M ammonium sulphate as precipitant, usingthe sitting drop method. Crystals were cryoprotected by transferringcrystals first into 10% (v/v) glycerol-precipitant mix before leaving in20% (v/v) glycerol;-precipitant. Diffraction data extending to 2.5 Åwere collected on beamline BM-14 of the ESRF. The structure was solvedusing molecular replacement program PHASER (18) which found three CBM40monomers in the asymmetric unit. Refinement was carried out using REFMAC(19) with COOT being used for model building (20). Data collection andrefinement statistics are shown in Table 3.

Isothermal Titration Calorimetry (ITC)

ITC experiments were performed on a VP-ITC microcalorimeter fromMicrocal Inc. (Northampton, Mass.) with a cell volume of 1.4 ml. Unlessstated otherwise, all ITC titrations were performed at 25° C. in 10 mMHEPES buffer pH7.4, containing 0.15M NaCl. For the characterization ofthe isolated CBM40 (1CBM), the following ligands were used:3′sialyllactose (3′SL), 6′sialyllactose, (6′SL), anddisialyl-lacto-N-tetraose (DSLNT), purchased from Dextra Labs (Reading,UK) and disialyllactose (DSL) from Sigma-Aldrich, UK (FIG. 2). Thelyophilised sialoside ligands were resuspended in degassed, filteredbuffer that was used for the dialysis of the peptide construct. For allother polypeptide constructs, 3′sialyllactose was used as the ligandthroughout. Protein concentrations were determined at A₂₈₀, usingcalculated molar extinction coefficients for 1CBM (38410 M⁻¹ cm⁻¹), 2CBMconstructs (75860 M⁻¹ cm⁻¹), 3CBM constructs (113790 M⁻¹ cm⁻¹) and4CBM(5) (151720 M⁻¹ cm⁻¹), respectively. The concentrations of CBM40polypeptides were 0.007-0.084 mM and the sialosides were 0.45-2.14 mM.Aliquots of sialosides (10 μl, unless stated otherwise) were titratedinto each polypeptide solution. The heats of dilution were subtractedfrom binding isotherm data before data were fitted by means of anonlinear regression analysis using a one-binding site model fromMicroCal Origin software.

Surface Plasmon Resonance (SPR)

Binding kinetics were determined by SPR using a BIACORE 3000 biosensorinstrument (GE Biosystems). Prior to use, a streptavidin coated (SA)biosensor chip was docked into the instrument and preconditioned withthree consecutive 1-minute injections of 1M NaCl in 50 mM NaOH.Biotinylated 3′sialyllactose-PAA (Glycotech) was diluted to 1 μg/ml inHBS-P (10 mM HEPES pH 7.4, 0.15M NaCl and 0.005% surfactant P20) beforebeing injected over 3 of the 4 flow cells in the chip. Typicalimmobilization levels of the ligand for each cell were approximately500RU. A reference surface was also prepared for subtraction of bulkeffects and non-specific interactions with streptavidin. The runningbuffer consisted of 10 mM HEPES pH 7.4 at a flow rate of 100 μl/min.

Interaction analysis for each peptide construct with immobilized3′sialyllactose was performed in running buffer at 15, 25 and 35° C.Purified peptide constructs were diluted into HBS-P to give a series ofconcentrations for 1CBM, 0.625 μM, 1.25 μM 2.5 μM, 5 μM, 10 μM; 2CBMconstructs 20 nM, 125 nM, 250 nM, 500 nM and 1000 nM; 3CBM constructs 1nM, 5 nM, 20 nM, 62.5 nM and 125 nM, and for 4CBM 0.18 nM, 0.5 nM, 1.6nM 5 nM and 15 nM. All analytes were injected over the flow cell surfaceat 30 μl/min. The dissociation of analyte from the surface was achievedin running buffer at the same flow rate for 3-5 min. Surfaces wereregenerated with two consecutive 30 s injections of 10 mM glycine-HCl pH2.5 at 30 μl/min. The affinity, as described by the equilibriumdissociation constant (K_(D)) was determined globally by fitting to thekinetic simultaneous ka/kd model, assuming Langmuir (1:1) binding, usingBIAevaluation software (BIAcore).

The free energy change of the interaction of the different CBMpolypeptide constructs with biotinylated 3′sialyllactose-PAA wasdetermined by using the equilibrium dissociation constants provided bythe ratios of the kinetic rate constants for each temperature. van'tHoff plots of ln K_(D) versus 1/T yielded values for ΔH/R from the slopeand −ΔS/R from the y intercept.

Plaque Assay

A virus plaque assay was used to demonstrate the efficacy of bindingmultivalent CBM40 polypeptides in the presence of the influenza A virus.Confluent (10⁶ cells) MDCK monolayers in 6-well plates were washed twicewith serum-free DMEM and incubated with either 1 ml solution of 2CBM40,or 3CBM40 (10 mg/ml, 10-fold serially diluted in serum-free DMEM) at 37°C. (+5% CO₂) for 1 h. Serum-free DMEM alone was used in parallel ‘mock’incubations. CBM40 reagents were then removed and monolayers wereinoculated with ˜80 PFU of influenza virus A/Udorn/72 [H3N2] for 1 h at37° C. (+5% CO2). Following virus adsorption, the inoculum was removedand cells were overlayed with 10 mM HEPES (pH 7.4) in DMEM supplementedwith 2 μg/ml trypsin and 1% (w/v) agarose. Inverted plates wereincubated at 37° C. (+5% CO2) for 2-3 days. Plaques were visualised byfixing the monolayers in 5% formaldehyde and staining with 0.1% crystalviolet.

Haemagglutination Assay

Chicken red blood cells in Alsever's solution were spun and washed atleast 4 times with PBS. The cells were finally resuspended in 1% (v/v)PBS. The vaccine strain X31 (with cell surface Hong Kong/68 HA and NA),was titrated in 96-well plates using a 2-fold serial dilution of a1:4000 stock in PBS, to which an equal volume of red blood cells hasbeen added. Plates were incubated at room temperature for at least 30mins. The endpoint was calculated as the lowest concentration of viruswhere hemagglutination is observed. CBMs were diluted appropriately andtitrated in PBS, to which an equal volume of cells has been added. Anendpoint for CBM binding to cells was also calculated. Experiments werethen carried out to determine the effect of a fixed concentration of1CBM with titrated virus versus virus only when incubated with chickenRBCs.

Development of CBM.TD Using an Oligomerisation Domain

The pEHISTEV construct containing 1CBM40, p1CBM, was modified to includean oligomerization domain at the C-terminus to allow the polypeptide toself-assemble into an oligomeric structure, when expressed in E. coli.For this, the trimerization domain from Pseudomonas aeruginosapseudominidase was used (Xu et al, 2009). The DNA fragment encodingresidues 333-440 was amplified by PCR using primers containing BamHI andXhoI and ligated to p1CNM, previously digested with the same enzymes.This was subsequently transformed into E. coli BL21(DE3) Gold cellsusing conditions similar to the growth and expression of all other CBM40constructs. The oligomer was purified using nickel affinity, and TEVprotease digestion, and presented a molecular weight of approximately100 kDa after gel filtration (data not shown).

Development of GFP-3CBM40 to Monitor Cell-Surface Bound Sialic Acid byCBM40

For the construction of a GFP-fused 3CBM40 fragment, the 3CBM40 fragmentwas digested with NcoI-XhoI from p3CBM(5) and inserted into a similarlydigested pEHISTEV vector containing the gene encoding the enhanced greenfluorescent protein (eGFP) downstream of the N-terminal Histag (Liu etal 2009; Connaris et al, 2009). This construct was tested against MDCKcells to determine whether the CBM40 bound to sialic acid at the cellsurface. For this, confluent monolayers of MDCK cells in 96-well plateswere incubated for 1 h at 37° C. with different concentrations ofGFP-3CBM before the addition of the influenza strain A/Udorn/72 (H3N2)virus (moi=0.002) for a further 1 h at the same temperature. The mixturewas removed and DMEM, without FCS, was added and plates were incubatedfor 72 hr.

Mouse Studies: Dose Finding Experiment:

To evaluate the prophylactic/therapeutic potential of CBM polypeptidesas an antiviral against the influenza virus, initial studies wereperformed to determine the dose of CBM40 required to administer in mice.These studies were conducted at the animal testing facility forinfluenza research at the Centre for Infectious Disease at Edinburgh.For this, BALB/c mice, 5-6 weeks of age, were used to perform a dosefinding experiment to test for adverse reactions, based on dosesinitially for the in vitro findings. Mice were dosed intranasally using3CBM40 polypeptide at 50 μg in 40 μl PBS, and at 500 μg in 40 μl PBS.PBS and BSA were used as controls. The mice were monitored daily forclinical signs and weighed daily from day 2 onwards. The mice wereculled on day 5 (except two of the 500 μg dosed mice, which were leftfor 14 days before culling).

Efficacy of CBM40 Against the Influenza A Virus

To evaluate the prophylactic and therapeutic efficacy of CBM40 againstinfluenza A/WSN/33(H1N1), BALB/c mice were lightly anaesthetised with aHalothane Oxygen mix before treatment with 3CBM40 and virus. The CBM40dosage for the experiment was 500 μg of 3CBM40 in PBS (40 μl), which wasdelivered intranasally, only once. BSA and PBS were used as thecontrols. Mice were treated with the CBM40 or the placebo, starting 1day before or on the day, or +1 or +3 days after intranasal infectionwith 10³ A/WSN/33 pfu. Virus titres for each group were determined 6days post infection where mice were culled and lungs homogenized in PBSto extract virus for plaque assays. Body weights of culled mice werealso measured to determine weight loss.

Results Structure of the Isolated CBM40 Module

The gene fragment encoding the sialic acid binding module CBM40 from theVibrio cholerae sialidase was subcloned into pEHISTEV and expressed inE. coli to generate 1CBM which was subsequently purified for binding andstructural studies. Initially, 1CBM was expressed insolubly albeit atvery high levels, but a heat shock of cultures at 42° C. during logphase of growth improved the solubility of this CBM such that up to50-70 milligram quantities per litre were produced consistently.Crystals of V. cholerae CBM40 were grown in the presence of3′sialyllactose. The asymmetric unit contained three CBM40 monomers, andeach had clear electron density for all three sugar moieties of theligand (FIG. 3A). Only sialic acid makes interactions with the protein,and these are the same as those made by sialic acid alone in complexwith the whole V. cholerae sialidase. There are three CBM40 modules inthe asymmetric unit of the crystal, with monomers A and B burying aninterface of some 750 Å² (FIG. 3B).

Determination of Sialoside and Linkage Specificity of V. cholerae CBM40

Studies were performed with 1CBM to determine its specificity towardsdifferent mono- and divalent-sialosides. Using ITC, the binding constantK_(a) and changes in enthalpy (ΔH) and entropy (ΔS) were measureddirectly for each interaction and stoichiometries (n, number of bindingsites) were determined from nonlinear least squares fit of the data tothe one-site binding model as described above. Data obtained from heatof dilution-corrected binding isotherms demonstrate the preference of1CBM for α-sialic acids (FIG. 4), exhibiting broad binding specificityto α(2,3), α(2,6) and α(2,8)-linked sialosides. All sialosides testeddisplayed similar affinity with dissociation constants, K_(d), rangingbetween 10-19 μM (at 25° C.) (Table 4, FIG. 4). The disialosides, DSLNTand DSL show 1.5-2 times greater affinity to sialic acid compared tomonovalent ligands. DSLNT showed a large change in enthalpy, with a ΔHvalue of −24 kcal/mol, compared to DSL (−12.5 kcal/mol). When comparingthe stoichiometries for binding DSLNT and DSL, corresponding n values of0.57 and 0.92 were observed, indicating that for the DSLNT, twomolecules of 1CBM are required to bind to 1 molecule of DSLNT, whereasfor DSL there is a 1:1 interaction. The difference here is due, not justto the number of sialic acid moieties present, but to their positionwithin the sialoside. Structurally, DSLNT is a branched divalentsialoside, unlike DSL, which has two sialic acid moieties linkedtogether in a linear fashion, so that only the terminal sialic acidmoiety is recognized (FIG. 2). This result indicates that the ΔH valueof DSLNT is approximately the sum of the individual binding enthalpiesof 3′sialyllactose, 6′sialyllactose and DSL, which have a stoichiometryof one.

Engineering of Multivalent, Sialic Acid-Specific CBM40 Polypeptides

We wanted to investigate whether sialic acid binding would occur ifidentical copies of 1CBM were linked together to produce a multivalentspecies. For this, copies of the gene encoding CBM40 were tetheredtogether with a DNA linker (representing up to 15 amino acids) to createpolypeptides of 2, 3 and 4 modules in tandem, which have been designatedas 2CBM(5), 2CBM(10), 2CBM(15), 3CBM(5), 3CBM(10) and 4CBM(5),respectively (the figure in parenthesis indicates the number of linkeramino acids) (FIG. 1B). Expression of these gene constructs wasperformed in E. coli and all demonstrated insolubility until cultureswere subjected to heat shock as for the isolated 1CBM. Afterpurification with nickel affinity chromatography, SDS PAGE analysis ofall peptide constructs demonstrated monomeric molecular weights of ˜21kDa, ˜42 kDa, ˜63 kDa and ˜85 kDa for 1CBM, 2CBM, 3CBM and 4CBMconstructs, respectively (data not shown).

The binding isotherms of the various engineered CBM40 polypeptides withmonovalent 3′sialyllactose are shown in FIG. 5. Using ITC, it wasrevealed that the binding of this sialoside to the designed CBM40polypeptides is enthalpically driven, with ΔH values being very similarranging from −12.3 to −16.3 kcal/mol at 25° C. (Table 5). There is alsovery little difference in all of the other thermodynamic parametersmeasured for each polypeptide interaction. In fact, the binding affinityof the multivalent CBM40s to sialic acid appeared to be similar to thatof the 1CBM. Furthermore, the length of the linker between modules wasnot shown, thermodynamically, to contribute significantly to thisinteraction (Table 5). Based on the one-site binding model, the n valuesdemonstrate the appropriate number of sites for each CBM40 polypeptide,interacting with 3′sialyllactose. The fact that no significant increasein affinity is seen as we increase the number of linked modules,suggests that the sialic acid-multivalent polypeptide interaction issimilar to that of a monovalent-monomeric one, indicating a simplebimolecular association.

Enhanced Binding Affinity of Multivalent CBM40 Polypeptides forMultivalent 3′ Sialyllactose

In order to test whether an avidity effect for sialic acid can beachieved with multivalent CBM40 polypeptides, surface plasmon resonance(SPR) was performed using biotinylated 3′sialyllactose immobilized on astreptavidin chip. Sensorgrams for all the CBM40 polypeptides injectedover immobilised 3′sialyllactose are shown in FIG. 6. The affinity foreach CBM40-3′SL interaction, described here as the equilibriumdissociation constant K_(d), was determined by a global fit modelderived from the ratio of association/dissociation rate constants(k_(a)/k_(d)), assuming Langmuir 1:1 binding (Table 6). An increase inaffinity towards sialic acid is observed as the number of modules isincreased. For the 1CBM-3′SL interaction at 25° C., there is a 10-foldincrease in binding (K_(d)˜1.8 μM) compared to that of the correspondingmonomeric-monovalent interaction (K_(d)˜18 μM) measured by ITC. Enhancedaffinity is observed when the number of modules increases to two, wherethere is an approximate 400-500-fold increase in binding, resulting inaffinities between 38-45 nM at 25° C. (Table 6). The linker lengthappears to influence marginally the avidity in this case, as only a1.2-fold increase in affinity is seen when increasing the number ofamino acids from 5 to 15. With three and four CBM40 modules there is afurther 10 to 20-fold enhancement in affinity to sialic acid, reachingaffinities of around 4 nM for the 5aa-, and 10aa-linked 3CBM modules,and 2.6 nM for the 4CBM module at 25° C. The highest affinity was4CBM(5) with a K_(d)˜861 pM when binding multivalent 3′SL at 15° C.Thus, it appears that a 7000-10000-fold increase in affinity can beachieved by going from a monovalent-monomeric interaction to amultivalent one, depending on the temperature of the interaction. Dataderived from van't Hoff plots for each CBM40 polypeptide-sialic acidinteraction measured at 15, 25 and 35° C. (FIG. 7, Table 7), demonstrateΔG values of −7.8 kcal/mol for 1CBM, around −10 kcal/mol for 2CBM, −11.3kcal/mol for 3CBM and −12 kcal/mol for 4CBM. The large difference in ΔGvalues between 1CBM and 2CBM, is also reflected in the changes inenthalpy and entropy of the interaction. Out of all the CBM40polypeptides, the 2CBM polypeptides gave the largest enthalpic changewith a ΔH value around −20 kcal/mol, which compensated a largeunfavourable entropic contribution (Table 7). This large difference inthe energetics between 1CBM and 2CBM interactions is also shown in theenhanced affinity of 2CBM to sialic acid, compared to 1CBM, 3CBM and4CBM polypeptides, suggesting cooperativety of the ligand-receptorinteraction.

In contrast, the 3CBM polypeptides showed small favourable entropicgains but the change in free energy of the interaction was stillstrongly negative due to a better favourable enthalpic penalty (Table7). Interestingly, both 3CBM and 4CBM polypeptides demonstrated lessnegative contributions to both ΔH and TΔS on ligand binding compared to1CBM and 2CBM polypeptides, despite the fact that free energy of theinteractions increased with increasing number of modules, which probablycontributed to the gain in affinities. This observation could be basedon a number of factors such as the interaction is sensitive to linkerflexibility due to the conformational arrangement of modules, theaccessibility of ligand binding sites, the modes of binding such asintra-, and intermolecular binding, and internal structural packing ofthe modules. The influence of linker length between the 3CBMpolypeptides, however, was negligible in terms of binding energy andaffinity, similar to the different 2CBM polypeptides. Since no relevantgain in affinity was achieved with 4CBM(5) at 25° C., it was decidedthat no further design of polypeptides would be undertaken.

Hemagglutination Assay

In order to test whether the monomeric and multivalent CBM40s bound tored blood cells and prevent binding of influenza virus, initialexperiments were performed with titrated CBMs, and with the titratedvirus, vaccine strain X31, against red blood cells. Preliminary resultsindicated that in the case of 1CBM40, no agglutination was seen, and asthis CBM is monomeric, it is expected to bind in a univalent fashion tocell surface sialic acids, ie a 1:1 association (FIG. 9 a). As for themultivalent CBM40s, both 2 and 3CBMs agglutinated red blood cells, asdid the vaccine X31 (FIG. 9 a). In addition, the end-point concentrationof the multivalent CBM required to agglutinate cells, was lower for3CBM40 than for the 2CBM (0.122 μM and 0.48 μM, respectively) suggestingthat an increase in the number of repeat modules increases the bindingaffinity of these to sialic acid. However, since the multivalent CBMsagglutinated red blood cells, it was decided to test a mixture oftitrated vaccine with a fixed concentration of 1CBM (71.25 μM) againstvaccine only to observe agglutination. As seen in FIG. 9 b, the presenceof 1CBM when challenged with vaccine, prevents binding of the virus,when compared to mock cells (virus only). When plates were left for 2.5days at 4° C., no agglutination was observed in the presence of 1CBM andvirus, suggesting the effectiveness of this CBM40 to blocking thebinding of a competing molecule.

Plaque Assay

Preliminary results of the plaque assay provide strong evidence tosuggest that the 1/100 dilution of 10 mg/ml 3CBM(5) is more efficient atblocking viral entry, into cells than the same diluted concentration of2CBM(10) (See FIG. 8). In particular, it can be seen that there is adifference in the number of plaques after incubation of MDCK cells withthe different repeat CBMs, when compared to mock cells, and when 3CBM(5)is used, in the presence of H3N2, very few plaques appear.

For 3CBM(5) ( 1/100 dilution), there is an 80-85% reduction of H3N2virus plaque forming units. For the 2CBM(10) result, although there isan effect on viral blocking, the same dilutions of the sameconcentration did not have the same effect as 3CBM(5). As such, itappears that an increase in number of CBM modules could be important ineffectively blocking viral binding to sialic acid at the cell surface.

Plaque Studies (Using CBMTD)

Virus infected MDCK cells:—the influenza strain A/Udorn/72 (H3N2) viruswas blocked by CBM40 polypeptides. FIG. 10 shows the cell protectioneffect of polypeptides 3CBM40, 4CBM40 and CBMTD on MDCK cells againstthe Udorn virus. There appears to be an increase in potency of CBMinhibition against the influenza strain as the number of linked CBM40sincrease from 3 to 4CBM40. Also, incubation of MDCK cells with theoligomer CBM.TD, before the addition of virus, showed effectiveinhibition at 0.5 mg/ml and significant reduction in the number ofplaques at 0.1 mg/ml CBM compared to the virus control (FIG. 10). Thesedata demonstrate that the V. cholerae sialidase CBM40, whethertandem-linked or as a self-assembled oligomer, of up to 4 domainrepeats, can effectively protect MDCK cells from virus infection.

Monitoring Cell-Surface Bound Sialic Acid by CBM40

FIG. 11 a shows the binding of GFP-3CBM40 to cell surface of MDCK cells1 h after the addition of virus. FIG. 11 b demonstrates that GFP-3CBM40does confer some protection from virus infection, even after 72 h postinfection when compared to the virus only well. Panel (a) alsodemonstrated that CBM (0.5 mg/ml) added to MDCK cells, in the absence ofvirus, showed no toxic effects after 72 h as cells were observed to beviable, with similar morphology to the uninfected control [panel (h)].

Mouse Studies (Dose Finding Experiment)

Table 1 shows the results of the starting and finishing weights of themice in the study. From clinical observations, the mice all appeared tobe very healthy, and that they all gained weight with no adverse effectsto 3CBM40 during the specified time period of 5 days and 14 days.Further studies, in terms of antibody response and lung tissuehistology, have yet to be carried out.

Efficacy of CBM40 Against Influenza A Virus

Preliminary results suggest that the administration of 3CBM40 one daybefore WSN virus challenge was shown to significantly reduce the effectsof viral infection in mice, demonstrating that CBM40 is effective as aprophylactic against the influenza A virus. This was evident in thereduction of viral titres of mice culled on day 6 p.i., compared to theBSA and PBS controls, where only an increase of 1.5 log titres wasmeasured from an initial dose of 10³ virus pfu (FIG. 12A). The mice inthis group also gained weight and showed no adverse effects from thevirus challenge (FIG. 12B). When 3CBM40 was administered at the sametime as the virus (Day 0), a 2.4-log increase of virus was observed 6days p.i, with weight gain being more than 100%, indicating that,despite the increase in viral titres, the mice tolerated the virusinfection. However, when CBM40 was administered 1 and 3 days after virusinfection, the mice appeared to lose weight and the viral titresincreased by 3 to 3.25 logs compared to the initial load, suggestingthat CBM40 may not be effective when given as a therapeutic, at leastunder the conditions described in the method. Further studies, whichwould include daily dosing during infection, instead of one dose, andoptimization of route administration, may determine whether CBM40 couldbe considered as a therapeutic antiviral, particularly in the earlystages of influenza infection. As a prophylactic, this protein biologiccould be delivered intranasally to protect the respiratory epithelialmucosa from virus attack.

Discussion

We have shown that the sialic acid-specific CBM from V. choleraesialidase can be successfully isolated from its parent enzyme andexploited to generate multivalent polypeptides that bind sialic acidwith high affinity. Using a combination of ITC and SPR, we have alsodemonstrated thermodynamics of binding of both monovalent andmultivalent forms of V. cholerae CBM40 to sialic acid, which appears inthe mono- and divalent form to be enthalpically driven, as are manyCBM-carbohydrate interactions to date (Boraston et al 2004, etc), butentropically driven when number of linked modules is greater than 2 whenintroduced to a multivalent surface.

With respect to its ligand and linkage specificity, the isolated CBM40preferentially binds α(2,3), α(2,6) and α(2,8)-linked sialic acids withmicromolar affinity. This binding affinity is similar to that reportedfor V. cholerae sialidase interaction with a non-hydrolysablethiosialoside (16). The fact that there is little discrimination betweendifferent linked sialosides can be seen in the crystal structure of theCBM40 complexed with 3′sialyllactose. The structure reveals an extensivenetwork of intermolecular interactions with sialic acid. The galactoseand glucose moieties do not interact with the protein domain, and thisis reflected in the similar K_(d) values and free energy of binding seenhere with different linked sialosides.

By exploiting the relatively high monovalent affinity of V. choleraeCBM40 for sialic acid, we have successfully engineered tandem-linkedrepeat polypeptides up to n=4 to achieve sub-nanomolar avidity wheninteracting with a multivalent surface. This is possibly the firstreport of a CBM that has been manipulated by fusing identical copies ofit using different length linker peptides. There have been previousreports of the use of isolated CBMs as molecular probes for analysis ofplant cell wall polymers (21) as well as the study of CBMs naturallyfound in multimodular glycoside hydrolases, which have been isolatedeither as separate modules or as combinations with their neighbouringmodules to determine their function (14,17,22).

We wanted to explore the thermodynamics of a multivalent interactionwith its receptor to determine whether conformational constraints wouldprevent these engineered polypeptides from binding. It is well knownthat linker length between protein subunits can influence thethermodynamics of an interaction, and in many cases, covalently linkingsubunits can enhance oligomeric stability of an interaction by reducingthe entropic driving force for dissociation (23). We engineered flexiblepeptide linkers between CBM modules ranging from 5 amino acids to 15amino acids to determine whether linker length would significantlyenhance the affinity of the engineered CBM polypeptides for sialic acid.In the case for 2-tandem linked CBMs, increasing the peptide linker to10 amino acids gave a significant reduction in conformational entropydespite observing a 1.5-2-fold increase in affinity to sialic acidbetween the different 2CBM constructs. This suggests that some degree offlexibility of the modules has occurred when two CBMs are linkedtogether. The binding interaction of 2CBMs to sialic acid also showed anincrease in binding enthalpy which compensated the large unfavourableentropic contribution, resulting in an increase of free energy ofbinding as well as enhanced affinity for sialic acid, when compared to1CBM-multivalent 3SL interaction. This suggests that binding of 2CBM tosialic acid is a very stable, intramolecular interaction and as for1CBM, is enthalpically driven. However, for 3CBMs interacting withimmobilised multivalent 3SL, there was no significant difference betweendifferent length peptides. In fact, entropy data derived from van't Hoffplots of the different 3CBM-sialic acid interactions all show anincrease in entropy. Also, a significant decrease in binding enthalpy,and a 10-fold enhancement of affinity was observed when going from 2 to3 CBMs. It is likely that the enhancement of affinity observed in thecase of 3CBM binding to sialic acid could result from the probableformation of stable aggregates occurring at the surface of the SPR chipand that the large change in entropy could be due to structurallyordered water molecules being released from the hydration shells,influenced by tight protein-protein packing. This can be seen from thestructure of the 1CBM-3SL complex, where three 1CBM monomers are closelypacked together (FIG. 3B). Entropy-driven interactions have been seen inother multivalent protein-carbohydrate systems particularly whereintermolecular binding occurs, leading to the formation of aggregates(24) (25), (26). Interestingly, increasing the number of linked modulesto 4, demonstrated a less favourable entropic contribution to that of3CBMs and favourable binding enthalpy although this is slightly lessthan that of the 1CBM interaction with immobilised 3′SL. The free energyof binding was slightly greater than all the other CBMs corresponding toa slight increase in affinity from 3 to 4CBM. It is likely that thevalency of the engineered tandem-linked polypeptides may play animportant part in the stabilization of oligomers and their interaction.Poon (20) describes that increasing the valency of a tandem-linkedsingle polypeptide chain can lead to a corresponding reduction in themolecularity of a tethered oligomer which, thermodynamically speaking,becomes more stable. In practice, Poon states that a tandem of valencyhigher than, or not divisible by the molecularity of the oligomer willresult in cross-linked oligomers. Thus for a tetrameric oligomer, suchas in the case of 4CBM, a tandem dimer or tetramer would be desirable,whereas a trimeric or pentameric oligomer requires a tandem of the samerespective valency.

These studies have shown that a CBM40 can be isolated and manipulated byfusing identical copies of it to enhance its affinity to sialic acidthrough avidity. Furthermore, we have shown that the monomer andengineered multimeric CBM40 from V. cholerae sialidase can be used aspotential antivirals against the influenza A virus. Using this type oftechnology, it is therefore possible that other CBMs could be isolatedand engineered for use as high affinity tools for glycan screening andprofiling. In addition, determination of a CBM structure in complex withits glycan can aid in the development of selective reagents for use inthe field of glycomics.

REFERENCES

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TABLE 1 Dose finding experiment of 3CBM40 administered in BALB/c mice.Mouse number Cage/group Dose Weight day 0 End weight 89 5 dys  50 ug3CBM 17.1 17.5 90 5 dys  50 ug 3CBM 15.05 15.5 91 5 dys PBS 12.5 13.1 925 dys PBS 14.7 15.4 93 5 dys 500 ug 3CBM 15.45 16.70 94 14 dys  500 ug3CBM 14.6 18.3 95 14 dys  500 ug 3CBM 14 18.1 96 5 dys 500 ug 3CBM 14.7516.05 97 5 dys 500 ug BSA 14.8 15.4 98 5 dys 500 ug BSA 13.4 14 99 Tkip16.4 16.60 100 Tkip 16.05 17.2 101 Tkip 16.45 18.6 102 Tkip 15 18.1 1030.5% DMSO 17.15 18.25 104 0.5% DMSO 14.85 16.8

TABLE 2 Oligonucleotide primers used to amplify DNA fragments. PrimerOligonucleotide sequence 1F CGTCCCATGGCACTTTTTGACTATAACGC(NcoI) 1RCCGGCTCGAGCTAGTCGCCTTGAATTTCAA AC(XhoI) 2F (5)CGTCGGATCCATGGCACTTTTTGAC(BamHI) 2R (5) GCACGGATCCGTTCAGGGCGTCGCCTTGAATTT(BamHI) 2F (10) GCCTGGATCCGGTATGGCACTTTTTGACTAT AAC(BamHI) 2R (10)GACCGGATCCACCTCCTGATCCGTTCAGGGCGTCG CC(BamHI) 2R (15)GACCGGATCCACCTCCACCTGATCCACCTCCTGAT CC(BamHI) 3F (5)CTGCAAGCTTTGGGAATGGCACTTTTTG AC(HindIII) 3R (5)GCACTTCCAAAGCTTGCAGGTCGCCTTGAATTT C(HindIII) 3F (10)GGTGGAAGCTTGATGGCACTTTTTGACTATA AC(HindIII) 3R (10)GTCCAAGCTTCCACCTCCTCCCAATGCTTGCAGGTCG CC(HindIII) 4F (5)GGTAGGGAATTCGGGAATGGCACTTTTTGACTATA AC(EcoRI) 4R (5)GCACTCCCGAATTCCCTCCGTCGCCTTGAAT TTC(EcoRI)

TABLE 3 X-ray data collection and refinement statistics. Numbers inparentheses refer to the highest resolution shell. Data collection Spacegroup C222₁ Unit cell edges (Å) a == 138.6, b = 197.6, c = 83.0 X-raysource ESRF BM-14 Resolution range 30-2.5 Å Completeness (%) 95.4 (88.6)R_(merge) 0.064 (0.414) <I/σI> 15.8 (2.7)  Refinement No. of reflectionswork/test 35,893/1,911 R_(cryst) 0.224 R_(free) 0.263 r.m.s.d. bonddistance (Å) 0.017 r.m.s.d bond angle (°) 1.716 R_(merge) =Σ_(hkl)Σ_(i)|I_(hkl, i) − <I_(hkl)>|/Σ_(hkl)<I_(hkl)> R_(cryst) andR_(free) = (Σ||F_(o)| − |F_(c)||)/(Σ|F_(o)|)

TABLE 4 Thermodynamic parameters of interaction of isolated CBM40 fromV. cholerae sialidase with different linkage sialosides. Linkage ΔH TΔSΔG K_(a) K_(d) Sialoside specificity n (kcal/mol) (kcal/mol) (kcal/mol)(10⁻⁴M) (μM) 3′SL α2, 3 0.96 ± 0.002 −16.3 ± 0.05  −9.8 −6.5 5.48 ± 0.0518 6′SL α2, 6 1.02 ± 0.001  −9.9 ± 0.002 −3.5 −6.4 5.16 ± 0.03 19 DSLα2, 8 α2, 3 0.92 ± 0.001 −12.5 ± 0.003 −5.7 −6.8 10.2 ± 0.01 9.8 DSLNTα2, 6, α2, 3 0.57 ± 0.001 −24.0 ± 0.007 −17.3 −6.7  7.8 ± 0.06 13

TABLE 5 ITC results of binding of CBM40 peptides to 3′sialyllactose(duplicate measurements). [P] [3′SL] ΔH TΔS ΔG K_(a) K_(d) Peptide mM mMn (kcal/mol) (kcal/mol) (kcal/mol) (10⁻⁴M) (μM) 1CBM 0.084 1.04 0.96 ±0.002 −16.3 ± 0.05 −9.8 −6.5 5.48 ± 0.05 18 2CBM(5) 0.071 2.19 2.00 ±0.003 −12.3 ± 0.03 −6.2 −6.1 3.18 ± 0.02 31 2CBM(10) 0.033 1.14 1.93 ±0.007 −13.7 ± 0.07 −7.3 −6.4 4.36 ± 0.05 22 2CBM(15) 0.026 0.79 1.99 ±0.014 −13.8 ± 0.01 −7.5 −6.3 3.49 ± 0.04 28 3CBM(5) 0.018 0.75 2.96 ±0.003 −13.5 ± 0.03 −7.2 −6.3 3.63 ± 0.04 27 3CBM(10) 0.016 0.72 3.09 ±0.024 −13.5 ± 0.01 −7.1 −6.4 4.54 ± 0.09 22 4CBM(5) 0.007 0.45 3.96 ±0.322 −15.8 ± 0.02 −9.6 −6.2 3.56 ± 0.03 28

TABLE 6 Biacore kinetic parameters for the different CBM polypeptidesinteracting with multivalent 3′sialyllactose (n = 3 determinations).Peptide T k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 1CBM 35  (3.1 ± 0.05) ×10³ (1.8 ± 0.3) × 10⁻² 5.8 × 10⁻⁶ 25 (4.3 ± 0.7) × 10³ (7.6 ± 0.4) ×10⁻³ 1.8 × 10⁻⁶ 15 (3.7 ± 0.4) × 10³ (3.6 ± 0.9) × 10⁻³ 9.7 × 10⁻⁷2CBM(5) 35 (3.5 ± 0.2) × 10⁵  (3.6 ± 0.04) × 10⁻² 1.0 × 10⁻⁷ 25 (5.6 ±0.7) × 10⁵ (2.5 ± 0.1) × 10⁻² 4.5 × 10⁻⁸ 15  (7.4 ± 0.13) × 10⁵  (9.5 ±0.07) × 10⁻³ 1.3 × 10⁻⁸ 2CBM(10) 35 (2.0 ± 0.1) × 10⁴ (2.6 ± 0.2) × 10⁻³1.3 × 10⁻⁷ 25 (5.9 ± 0.3) × 10⁵  (2.3 ± 0.23) × 10⁻² 3.9 × 10⁻⁸ 15  (8.1± 0.17) × 10⁵  (6.9 ± 0.05) × 10⁻³ 8.5 × 10⁻⁹ 2CBM(15) 35 (5.3 ± 0.6) ×10⁵ (5.2 ± 0.3) × 10⁻² 9.8 × 10⁻⁸ 25 (8.8 ± 0.5) × 10⁵  (3.4 ± 0.08) ×10⁻² 3.8 × 10⁻⁸ 15 (8.7 ± 0.5) × 10⁵ (8.7 ± 0.3) × 10⁻³  1 × 10⁻⁸3CBM(5) 35  (5.2 ± 0.35) × 10⁵ (4.4 ± 0.2) × 10⁻³ 8.44 × 10⁻⁹  25  (5.6± 0.15) × 10⁵  (2.3 ± 0.08) × 10⁻³ 4.0 × 10⁻⁹ 15 (3.0 ± 0.3) × 10⁵  (1.1± 0.04) × 10⁻³ 3.7 × 10⁻⁹ 3CBM(10) 35  (5.1 ± 0.01) × 10⁵  (4.1 ± 0.48)× 10⁻³ 8.07 × 10⁻⁹  25 (4.5 ± 0.4) × 10⁵ (1.95 ± 0.03) × 10⁻³ 4.26 ×10⁻⁹  15 (3.3 ± 0.2) × 10⁵ (1.21 ± 0.02) × 10⁻³ 3.63 × 10⁻⁹  4CBM(5) 35(2.2 ± 0.8) × 10⁵  (9.2 ± 0.07) × 10⁻⁴ 4.01 × 10⁻⁹  25 (2.8 ± 0.5) × 10⁵(7.4 ± 0.9) × 10⁻⁴ 2.62 × 10⁻⁹  15 (2.9 ± 0.4) × 10⁵  (2.5 ± 0.01) ×10⁻⁴ 8.61 × 10⁻¹⁰ 

TABLE 7 Thermodynamic parameters for the interaction of designed sialicacid binding peptide modules with multivalent 3′sialyllactose-PAA-biotin derived from van't Hoff plots. ΔG (kcal/mol) ΔH(kcal/mol)ΔS(cal/mol) TΔS(kcal/mol) 1CBM −7.8 −15.9 −27.3 −8.1 2CBM(5) −10.0 −18.8−29.4 −8.8 2CBM(10) −10.3 −22.4 −40.8 −12.1 2CBM(15) −10.1 −20.7 −35.4−10.6 3CBM(5) −11.3 −7.2 13.8 4.1 3CBM(10) −11.3 −7.1 14.2 4.2 4CBM(5)−11.9 −14.1 −7.5 −2.2

1-2. (canceled)
 3. A pharmaceutical composition comprising acarbohydrate binding molecule (CBM), multivalent CBM or CBM polymer anda pharmaceutically acceptable excipient, for use in treating orpreventing a disease and/or condition caused or contributed to by one ormore pathogens.
 4. The pharmaceutical composition of claim 3 formulatedfor oral, parenteral or topical administration.
 5. A method of treatingor preventing a disease caused or contributed to by a pathogen, saidmethod comprising the step of administering a therapeutically effectiveamount of a carbohydrate binding module (CBM).
 6. The method of claim 5,wherein the diseases or conditions are caused by viral respiratorypathogens and/or bacterial pathogens.
 7. The method of claim 5, whereinthe viral respiratory pathogen belongs to the Orthomyxoviridae orParamyxoviridae families.
 8. The method of claim 5, wherein thebacterial pathogens is of the Streptococcus genus, Haemophilusinfluenzae or Pseudomonas aeruginosa.
 9. The method of claim 5 whereinthe disease caused or contributed to by a pathogen is influenza, croup,pneumonia and/or bronchitis.
 10. The method of claim 5, wherein the CBMexhibits an affinity for, or binds to, sialic acid.
 11. The method ofclaim 5, wherein the CBM belongs to the family 40 CBMs.
 12. The methodof claim 5, wherein the CBM is derived from Vibrio cholerae orClostridium perfringens.
 13. The method of claim 5, wherein the CBM is amultivalent CBM comprising two or more CBM monomers.
 14. A multivalentcarbohydrate binding molecule (CBM) or CBM polymer comprising two ormore CBM molecules.
 15. The multivalent CBM or CBM polymer of claim 14,which binds to, or has affinity for, one or more cell surfacecarbohydrates.
 16. The multivalent CBM or CBM polymer of claim 14,wherein the two or more CBM molecules are the CBM40 molecules derivedfrom Vibrio cholerae or Clostridium perfringens.
 17. A method ofgenerating a carbohydrate binding molecule (CBM) polymer, said methodcomprising the steps of: (a) ligating CBM encoding nucleotide sequences;and (b) expressing the ligated CBM nucleotide sequences to generate aCBM polymer.
 18. A method of screening for or identifying carbohydratebinding molecules (CBM) potentially useful in the treatment of diseasesand/or conditions, said method comprising the steps of: (i) contacting aCBM or CBM polymer with a cell in the presence of a pathogen known tobind or infect said cell; (ii) identifying those cells to which thepathogen has bound or which the pathogen has infected; and (iii)comparing the results with a standard or control assay in which no CBMor CBM polymer has been added; wherein a decrease in the number of cellsto which the pathogen has bound or a decrease in the number of cellsinfected by the pathogen relative to the control assay, indicates a CBMor CBM polymer potentially useful in the treatment of a disease and/orcondition caused or contributed to by that pathogen.
 19. A method ofscreening, identifying, detecting, labeling and/or tagging acarbohydrate in a sample, said method comprising the step of: (a)contacting a sample with a carbohydrate binding molecule (CBM) or CBMpolymer, under conditions suitable to permit binding between the CBM/CBMpolymer and the carbohydrate(s) it/they bind or have affinity for; (b)removing unbound CBM or CBM polymer; and (c) detecting bound CBM or CBMpolymer.
 20. The method of claim 19, wherein the sample comprises cellsderived from a tissue biopsy, scraping or secretion.
 21. The compositionclaim 3, wherein the CBM or CBM polymer is modified to include a moietyto be delivered to a cell.
 22. The method of claim 5 wherein the CBM orCBM polymer is modified to include a moiety to be delivered to a cell.23. The composition of claim 14, wherein the multivalent CBM or CBMpolymer is modified to include a moiety to be delivered to a cell. 24.The method of claim 17, wherein the CBM polymer is further modified toinclude a moiety to be delivered to a cell.
 25. The pharmaceuticalcomposition of claim 3 wherein the CBM, multivalent CBM or CBM polymerbelongs to, or comprises, the family 40 CMBs.
 26. The pharmaceuticalcomposition of claim 3, wherein the CBM, multivalent CBM or CBM polymeris, or comprises CBM40 derived from Vibrio cholerae or Clostridiumperfringens.